Longevous cultures of aciduric bacteria



Patentecl July 11, 1933 UNITED STATES FERDINAND W. NITARDY, 0E"BROOKLYN,

NEW YORKQAND JOHN J. ENRIGHT AND VINCENT S. WRENN, OF PITTSBURGH,PENNSYLVANIA, TO 13. R. SQUIBB 85 SONS, (3F NEXV YORK, N. Y., ACORPORATION OF NEW YGBK LONGEVOUS CULTURES OF ACIDURIC BACTERIA NoDrawing.

and proliferation of aciduric bacteriafor.

example, Lactobacillus, particularly Lactobacillus a0icZ0p]vz'Zusin theintestinal tract is beneficial when pathogenic or putrefactive organismsare present, and exercises a favorable effect on various disorders,including constipation.

However, cultures hitherto designed and marketed for accomplishing thisimplantation have been characterized by the early death of the majorpart of their bacilli, owing to the high concentration of lactic acidproduced by them. Even in milk, authoritati'vely held to be the bestmedium, the microorganism are decimated with such rapidity that theircount is reduced to asmall percentage in a few days. As a consequence ofthis instability, a considerable economic waste has been suffered; anddaily distribution of fresh cultures has beenrendered necessary,

wherefore preparations of the fullest thera-' peutic value can besupplied only to communities situated within certain territorial limits.lVIoreover, the milk medium commonly employed for cultures is quicklysoured by the metabolic processes of the bacteria, and thus renderedextremely unpalatable to many who would otherwise avail themselves ofthe treatment. Broth cultures flavored with fruit juices are known, buttheir bacteria are so ephemeral that such cultures have to be consumedin volumes about thirty times as great as those of fresh milk culturesto obtain the same effective dose.

It is an object of our invention to provide longer-lived cultures ofaciduric bacteria than those hitherto known. A further object is toprovide such longevous cultures optionally in media, of unobjectionabletaste, other than milk. A still further object is to provide a methodwhereby these cultures may be prepared.

In the practice of our invention we maintain the cultures of aciduricbacteria in the presence of a bu'flerby which term we mean to includeall substances and/or conditions Application filed April 1 1931. Serial1 To..527,041.

that, withoutimpairing the viability of the micro-organisms, minimizechange in the hydrogen-ion concentration of the mediumand thus, byarresting the growth of active acidity, prevent the formation ofa lethalenvironment. o have found, for instance, that the application of analiphaticacid saltsay about one percent of an alkalimetal lactatewillaccomplish our purpose; cit'rates, gluconates, adipates, and malatesbeing among the other salts that may be used.

Furthermore, these aliphatic-acid salts have a strong anti-flocculentefiect: whereas the bacterial mass of liquid cultures would otherwiseagglomerate and settle out, the presence of the salt causes themicro-organisms to remain uniformly suspended for long periods.

A specific example of the practice of our invention is as follows: Apeptone-whey broth medium is first prepared by heating skimmed milk tobetween 85 and 90 C., enough ten-percent hydrochloric acid is added toprecipitate all the casein,the whey is separated by filtration throughseveral thicknesses of cheese-cloth, and, the reaction being adjusted topH 6.0 with ten-percent sodium hydroxide, the whey is placed in flasksplugged with cotton, which are autoclaved at twenty pounds for thirtyminutes; the lactalbumin settles, and after the supernatant whey isdecanted and filtered, 5.0 grams ofpeptone is added to the liter ofwhey, the reaction is adjusted to pH- 6.0, and sterilization is effected by autoclaving. Selection is made of an actively growing strain ofLaotobaozlluc acidophilus of the desired type, which is passed throughseveral successive transplants in sterile peptone-whey broth until aheavy seed ing culture can be obtained by three or four days incubation,when 100 cc. is transferred to the large flask containing one liter ofsterile peptone-whey broth; and incubated at 37 C. for 72 hours Then 25cc. quantities of the culture are aseptically transferred to sterile 220cc. bottles containing, sterile, 105 cc. of water, 40 cc. of 27.7 Baumsucrose syrup, 8 cc. of true-fruit strawberry extract, and 22 cc. of10.2% aqueous sodium lactate solution; and the bottles are sealed.

embody various types of micro-organisms,

culture media (including milk), buffers, and

processes, and may be employed in Various industrial fermentativeoperations.

e claim: 1. The method oi? prolonging the life of cultures ofLactobacillus suitable for intestinal implantation WlllCh comprisesmaintain ing them in the presence ofa'buil'er consistnal implantationwhich comprises maintaining of an aliphatic-acid salt.

'2. The method of prolonging the life of cultures of Lactobacillussuitable for intesti ing them in the presence of sodium lactate.

3. The method of prolonging the life of cultures of Lactobacillusacdophilussuitable for intestinal implantation which comprisesmaintaining them in the presense of a buffer consisting of analiphatic-acid salt.

4. ,The method of prolonging the life of cultures of Lactobacz'llusaoidoph-il zzs suitable" for intestinal implantation which comprisesmaintaining them in the presence of sodium lactate. r

5. Cultures of Lactobacillus suitable for intestinal implantationincluding a butler consisting of an aliphatic-acid salt.

6. Cultures of Lactobacillus suitable for intestinal implantationincluding sodium lac tate.

7. Cultures of Lactobacillus acz'clophz'lus suitable for intentinalimplantation includ.

ing a buffer consisting of an aliphatic-acid salt. a

8. Cultures of 'Lactobacillus aciclophilus suitable for intestinalimplantation including sodium lactate. I

9. Culturescof Lactobacilhbs acidophz'lus suitable for intestinalimplantation, in media other than milk, including sodium lactate.

In Witness whereof We afiix our signatures. l

FERDINAND V. NITARDY. JOHN J. ENRIGHII VINCENT S. vVRENN.

